Methods and Compositions for Treating Conditions of the Eye

ABSTRACT

Provided are methods and compositions for treating ocular conditions characterized by the presence of unwanted choroidal neovasculature, for example, neovascular age-related macular degeneration. The selectivity and sensitivity of, for example, a photodynamic therapy (PDT)-based approach can be enhanced by combining the PDT with an anti-FasL factor, for example, an anti-FasL neutralizing antibody.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of and priority to U.S.Patent Application Ser. No. 60/655,723, filed Feb. 23, 2005, the entiredisclosure of which is incorporated by reference herein for allpurposes.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions for treatingocular conditions and, more specifically, the invention relates tophotodynamic therapy-based methods and compositions for treating ocularconditions characterized by unwanted choroidal neovasculature.

BACKGROUND

Choroidal neovascularization can lead to hemorrhage and fibrosis, withresulting visual loss in a number of conditions of the eye, including,for example, age-related macular degeneration, ocular histoplasmosissyndrome, pathologic myopia, angioid streaks, idiopathic disorders,choroiditis, choroidal rupture, overlying choroid nevi, and certaininflammatory diseases. One of the disorders, namely, age-related maculardegeneration (AMD), is the leading cause of severe vision loss in peopleaged 65 and above (Bressler et al. (1988) Surv. Ophthalmol. 32, 375-413,Guyer el al. (1986) Arch. Ophthalmol. 104, 702-705, Hyman et al. (1983)Am. J. Epidemiol. 188, 816-824, Klein & Klein (1982) Arch. Ophthalmol.100, 571-573, Leibowitz et al. (1980) Surv. Ophthalmol. 24, 335-610).Although clinicopathologic descriptions have been made, little isunderstood about the etiology and pathogenesis of the disease.

Dry AMD is the more common form of the disease, characterized by drusen,pigmentary and atrophic changes in the macula, with slowly progressiveloss of central vision. Wet or neovascular AMD is characterized bysubretinal hemorrhage, fibrosis and fluid secondary to the formation ofchoroidal neovasculature (CNV), and more rapid and pronounced loss ofvision. While less common than dry AMD, neovascular AMD accounts for 80%of the severe vision loss due to AMD. Approximately 200,000 cases ofneovascular AMD are diagnosed yearly in the United States alone.

Currently, treatment of the dry form of age-related macular degenerationincludes administration of antioxidant vitamins and/or zinc. Treatmentof the wet form of age-related macular degeneration, however, has provedto be more difficult. Currently, two separate methods have been approvedin the United States of America for treating the wet form of age-relatedmacular degeneration. These include laser photocoagulation andphotodynamic therapy using a benzoporphyrin derivative photosensitizer.During laser photocoagulation, thermal laser light is used to heat andphotocoagulate the neovasculature of the choroid. A problem associatedwith this approach is that the laser light must pass through thephotoreceptor cells of the retina in order to photocoagulate the bloodvessels in the underlying choroid. As a result, this treatment destroysthe photoreceptor cells of the retina creating blind spots withassociated vision loss. During photodynamic therapy, a benzoporphyrinderivative photosensitizer is administered to the individual to betreated. Once the photosensitizer accumulates in the choroidalneovasculature, non-thermal light from a laser is applied to the regionto be treated, which activates the photosensitizer in that region. Theactivated photosensitizer generates free radicals that damage thevasculature in the vicinity of the photosensitizer (see, U.S. Pat. Nos.5,798,349 and 6,225,303). This approach is more selective than laserphotocoagulation and is less likely to result in blind spots. Undercertain circumstances, this treatment has been found to restore visionin patients afflicted with the disorder (see, U.S. Pat. Nos. 5,756,541and 5,910,510). During clinical studies, however, it has been found thatrecurrence of leakage appears in at least a portion of the CNV by one tothree months post-treatment. Increasing photosensitizer or light dosesdo not appear to prevent this recurrence, and can even lead to undesirednon-selective damage to retinal vessels (Miller et al. (1999) Archivesof Ophthalmology 117: 1161-1173). Another avenue of investigation is torepeat the PDT procedure over prolonged periods of time. The necessityfor repeated PDT treatments can nevertheless be expected to lead tocumulative damage to the retinal pigment epithelium (RPE) andchoriocapillaris, which may lead to progressive treatment-related visionloss. In addition, PDT can cause transient visual disturbances,injection-site adverse effects, transient photosensitivity reactions,infusion-related back pain, and vision loss.

Therefore, there is still a need for improved methods for treating AMDcharacterized by unwanted choroidal neovasculature that increase theefficacy and selectivity of treatment, and which reduce or delay arecurrence of the disorder.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions fortreating ocular conditions associated with unwanted choroidalneovasculature. Such conditions include, for example, neovascular AMD,ocular histoplasmosis syndrome, pathologic myopia, angioid streaks,idiopathic disorders, choroiditis, choroidal rupture, overlying choroidnevi, and certain inflammatory diseases. The invention, for example,provides a more effective PDT-based method for treating unwanted CNVthat has one or more of the following advantages: increased efficacy oftreatment; increased selectivity for CNV; and reduced or delayedrecurrence of the condition following PDT.

In one aspect, the invention provides a method of treating unwanted CNVin a mammal, wherein the CNV comprises endothelial cells, for example,capillary endothelial cells. The method comprises the steps of: (a)administering to the mammal, for example, a primate, preferably, ahuman, an anti-FasL factor in an amount sufficient to permit aneffective amount to localize in the CNV; (b) administering to the mammalan amount of a photosensitizer (PDT dye) sufficient to permit aneffective amount to localize in the CNV; and (c) irradiating the CNVwith laser light such that the light is absorbed by the photosensitizerso as to occlude the CNV. During practice of this method, the anti-FasLfactor can enhance the activity of PDT. For example, the anti-FasLfactor and the PDT may act synergistically.

A variety of anti-FasL factors may be used in the invention. Usefulanti-FasL factors, include, for example, anti-FasL neutralizing antibody(available, for example, from Pharmingen, San Diego, Calif.); peptidesand nucleic acids (for example, anti-FasL aptamers) that bind FasL toprevent or reduce its binding to its cognate receptor, certainantibodies and antigen binding fragments thereof and peptides that bindpreferentially to the Fas receptor; antisense nucleotides and doublestranded RNA for RNAi that ultimately reduce or eliminate the productionof either FasL or the Fas receptor; soluble Fas; soluble FasL; decoyreceptor-3 (DcR3) analogues; matrix metalloproteinases (MMPs);vasoactive intestinal peptide (VIP); pituitary adenylatecyclase-activating polypeptide (PACAP); forskolin; combined use ofbenazepril and valsartan; nonpeptidic corticotropin-releasing hormonereceptor type 1 (CRH-R1)-specific antagonists; mimosine; peptides thatproduce a defective Fas-FasL complex; platelet-activating factor (PAF);and endothelin-1 (ET-1). These anti-FasL factors can act as direct orindirect antagonists of FasL activity.

The term “antibody,” as used herein, includes, for example, a monoclonalantibody or an antigen binding fragment thereof (for example, an Fv,Fab, Fa′ or an (Fa′)₂ molecule), a polyclonal antibody or an antigenbinding fragment thereof, or a biosynthetic antibody binding site, forexample, an sFv (U.S. Pat. Nos. 5,091,513; 5,132,405; 5,258,498; and5,482,858) that binds specifically to a target ligand. As used herein,the terms binds “specifically” or “preferentially” are understood tomean that the targeting molecule, for example, the antibody, binds tothe complementary or target ligand with a binding affinity of at least10⁵ M⁻¹, and more preferably 10⁷ M⁻¹.

The anti-FasL factor may, under certain circumstances, beco-administered simultaneously with the photosensitizer. Alternatively,the anti-FasL factor may be administered before or after thephotosensitizer. In a preferred embodiment, however, the anti-FasLfactor is administered to the mammal prior to administration of thephotosensitizer.

In another aspect, the invention provides an improved method of treatingunwanted choroidal neovasculature in a mammal. The improvement includesadministering to the mammal an effective amount of an anti-FasL factorso as to relieve side effects associated with a method for treatingunwanted choroidal neovasculature. The anti-FasL factor can include ananti-FasL antibody, and the side effects can include photoreceptor celldeath. In certain instances, the anti-FasL factor reduces apoptotic celldeath of photoreceptor cells during the method of treating unwantedchoroidal neovasculature. The method of treating unwanted choroidalneovasculature can include photodynamic therapy using a benzoporphyrinderivative photosensitizer, can include administering an effectiveamount of an anti-VEGF aptamer, an effective amount of an anti-VEGFantibody, and/or an effective amount of an anti-VEGF siRNA; and/or caninclude ameliorating the symptoms of age-related macular degeneration.

In all the methods disclosed herein, it is contemplated that anyphotosensitizer useful in PDT may be useful in the practice of theinvention. Useful photosensitizers include, for example, amino acidderivatives, azo dyes, xanthene derivatives, chlorins, tetrapyrrolederivatives, phthalocyanines, and assorted other photosensitizers.Preferred photosensitizers, include, for example, lutetium texaphyrin,benzoporphyrin and derivatives thereof, and hematoporphyrin andderivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of the presentinvention, as well as the invention itself, may be more fully understoodfrom the following description of preferred embodiments, when readtogether with the accompanying drawings, in which:

FIG. 1 depicts a schematic drawing of the FasL apoptotic pathway;

FIG. 2 provides evidence that anti-FasL treatment prevents cleavage ofbid after PDT;

FIG. 3 provides evidence that PDT increases the expression of the Fasreceptor in the rat retina;

FIG. 4 provides evidence that anti-FasL treatment prevents activation ofcaspase 3 after PDT;

FIG. 5 provides evidence that anti-FasL treatment reduces angiographicleakage after PDT;

FIG. 6 provides evidence that anti-FasL treatment reduces angiographicleakage in laser-induced CNV;

FIG. 7 provides evidence that anti-FasL treatment reduces PDT-inducedcaspase 8 activation in laser-induced CNV;

FIG. 8 provides evidence that anti-FasL treatment reduces PDT-inducedcytochrome c release in laser-induced CNV;

FIG. 9 provides evidence that anti-FasL treatment reduces PDT-inducedBax upregulation in laser-induced CNV; and

FIG. 10 provides evidence that anti-FasL treatment reduces PDT-inducedBcl-2 downregulation in laser-induced CNV.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an improved method for treating ocularconditions characterized as having unwanted CNV. Such conditionsinclude, for example, neovascular AMD, ocular histoplasmosis syndrome,pathologic myopia, angioid streaks, idiopathic disorders, choroiditis,choroidal rupture, overlying choroid nevi, and certain inflammatorydiseases. The invention provides one or more of the followingadvantages: increased efficacy of treatment; increased selectivity forCNV; and reduced or delayed recurrence of the condition following PDT.

The invention provides an improved method for treating ocular disorders,for example, AMD, characterized by unwanted choroidal neovasculature.The improved method comprises administering to the mammal an effectiveamount of an anti-FasL factor, for example, an anti-FasL antibody, forpreserving photoreceptor viability, i.e., reducing collateral retinaldamage during a treatment of unwanted choroidal neovasculature. Forexample, the anti-FasL factor may be combined with an anti-VEGF aptamer,for example the Macugen® aptamer (see the URL addresseyetk.com/science/science_venf.asp), for treatment of AMD (availablefrom Eyetech Pharmaceuticals, Inc., NY, N.Y.). Alternatively, theanti-FasL factor may be combined with a VEGF specific RNAi for thetreatment of AMD (see the URL address:alnvlam.com/therapeutic-programs/programs.asp) (available from AlnylamPharmaceuticals, Cambridge, Mass.). Similarly, the anti-FasL factor maybe combined with an anti-VEGF antibody or antibody fragment for thetreatment of AMD (see the URL address:gene.com/gene/products/information/oncology/avastin/index.jsp)(available from Genentech, Inc., San Francisco, Calif.).

In one aspect, the invention provides an improved PDT-based method fortreating unwanted target CNV. The method involves administration of aphotosensitizer to a mammal in need of such treatment in an amountsufficient to permit an effective amount (i.e., an amount sufficient tofacilitate PDT) of the photosensitizer to localize in the target CNV.After administration of the photosensitizer, the CNV then is irradiatedwith laser light under conditions such that the light is absorbed by thephotosensitizer. The photosensitizer, when activated by the light,generates singlet oxygen and free radicals, for example, reactive oxygenspecies, that result in damage to surrounding tissue. For example,PDT-induced damage of endothelial cells results in platelet adhesion anddegranulation, leading to stasis and aggregation of blood cells andvascular occlusion.

An increase in efficacy and/or selectivity of the PDT, and/or reductionor delay of recurrence of the CNV, can be achieved by administering ananti-FasL factor to the mammal prior to, concurrent with, or afteradministration of the photosensitizer. It is contemplated that a varietyof photosensitizers useful in PDT may be useful in the practice of theinvention and include, for example, amino acid derivatives, azo dyes,xanthene derivatives, chlorins, tetrapyrrole derivatives,phthalocyanines, and assorted other photosensitizers.

Amino acid derivatives include, for example, 5-aminolevulinic acid (Berget al. (1997) Photochem. Photobiol. 65: 403-409; El-Far et al. (1985)Cell. Biochem. Function 3, 115-119). Azo dyes, include, for example,Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Black, Disperse Orange,Disperse Red, Oil Red O, Trypan Blue, Congo Red, β-carotene (Mosky etal. (1984) Exp. Res. 155, 389-396). Xanthene derivatives, include, forexample, rose bengal.

Chlorins include, for example, lysyl chlorin p6 (Berg et al. (1997)supra) and etiobenzochlorin (Berg et al. (1997) supra), 5, 10, 15,20-tetra (m-hydroxyphenyl) chlorin (M-THPC), N-aspartyl chlorin e6(Dougherty et al. (1998) J. Natl. Cancer Inst. 90: 889-905), andbacteriochlorin (Korbelik et al. (1992) J. Photochem. Photobiol. 12:107-119).

Tetrapyrrole derivatives include, for example, lutetium texaphrin(Lu-Tex, PCI-0123) (Dougherty et al. (1998) supra, Young et al. (1996)Photochem. Photobiol. 63: 892-897), benzoporphyrin derivative (BPD)(U.S. Pat. Nos. 5,171,749, 5,214,036, 5,283,255, and 5,798,349, Jori etal. (1990) Lasers Med. Sci. 5, 115-120), benzoporphyrin derivative monoacid (BPD-MA) (U.S. Pat. Nos. 5,171,749, 5,214,036, 5,283,255, and5,798,349, Berg et al. (1997) supra, Dougherty et al. (1998) supra),hematoporphyrin (Hp) (Jori et al. (1990) supra), hematoporphyrinderivatives (HpD) (Berg et al. (1997) supra, West et al. (1990) In. J.Radiat. Biol. 58: 145-156), porfimer sodium or Photofrin (PHP) (Berg etal. (1997) supra), Photofrin II (PII) (He et al. (1994) Photochem.Photobiol. 59: 468-473), protoporphyrin IX (PpIX) (Dougherty et al.(1998) supra, He et al. (1994) supra), meso-tetra (4-carboxyphenyl)porphine (TCPP) (Musser et at (1982) Res. Commun. Chem. Pathol.Pharmacol. 2, 251-259), meso-tetra (4-sulfonatophenyl) porphine (TSPP)(Musser et al. (1982) supra), uroporphyrin I (UROP-I) (El-Far et al.(1985) Cell. Biochem. Function 3, 115-119), uroporphyrin III (UROP-III)(El-Far et al. (1985) supra), tin ethyl etiopurpurin (SnET2), (Doughertyet al. (1998) supra 90: 889-905) and13,17-bis[1-carboxypropionyl]carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetranethyl6 porphyrin sodium (ATX-S10(Na)) Mori et al. (2000) JPN. J. CANCER RES.91:753-759, Obana et al. (2000) Arch. Ophthalmol. 118:650-658, Obana etal. (1999) Lasers Surg. Med. 24:209-222).

Phthalocyanines include, for example, chloroaluminum phthalocyanine(AlPcCI) (Rerko et al. (1992) Photochem. Photobiol. 55, 75-80), aluminumphthalocyanine with 2-4 sulfonate groups (AlPcS2-4) (Berg et al. (1997)supra, Glassberg et al. (1991) Lasers Surg. Med. 11, 432-439),chloro-aluminum sulfonated phthalocyanine (CASPc) (Roberts et al. (1991)J. Natl. Cancer Inst. 83, 18-32), phthalocyanine (PC) (Jori et al.(1990) supra), silicon phthalocyanine (Pc4) (He et al. (1998) Photochem.Photobiol. 67: 720-728, Jori et al. (1990) supra), magnesiumphthalocyanine (Mg2+-PC) (Jori et al. (1990) supra), and zincphthalocyanine (ZnPC) (Berg et al. (1997) supra). Other photosensitizersinclude, for example, thionin, toluidine blue, neutral red and azure c.

However, useful photosensitizers, include, for example, LutetiumTexaphyrin (Lu-Tex), a new generation photosensitizer having favorableclinical properties including absorption at about 730 nm permitting deeptissue penetration and rapid clearance. Lu-Tex is available from AlconLaboratories, Fort Worth, Tex. Other useful photosensitizers includebenzoporhyrin and benzoporphyrin derivatives, for example, BPD-MA andBPD-DA, available from QLT Inc., Vancouver, Canada.

The photosensitizer preferably is formulated into a delivery system thatdelivers high concentrations of the photosensitizer to the CNV. Suchformulations may include, for example, the combination of aphotosensitizer with a carrier that delivers higher concentrations ofthe photosensitizer to CNV and/or coupling the photosensitizer to aspecific binding ligand that binds preferentially to a specific cellsurface component of the CNV.

In one embodiment, the photosensitizer can be combined with a lipidbased carrier. For example, liposomal formulations have been found to beparticularly effective at delivering the photosensitizer, greenporphyrin, and more particularly BPD-MA to the low-density lipoproteincomponent of plasma, which in turn acts as a carrier to deliver thephotosensitizer more effectively to the CNV. Increased numbers of LDLreceptors have been shown to be associated with CNV, and by increasingthe partitioning of the photosensitizer into the lipoprotein phase ofthe blood, it may be delivered more efficiently to the CNV. Certainphotosensitizers, for example, green porphyrins, and in particularBPD-MA, interact strongly with lipoproteins. LDL itself can be used as acarrier, but LDL is more expensive and less practical than a liposomalformulation. LDL, or preferably liposomes, are thus preferred carriersfor the green porphyrins since green porphyrins strongly interact withlipoproteins and are easily packaged in liposomes. Compositions of greenporphyrins formulated as lipocomplexes, including liposomes, aredescribed, for example, in U.S. Pat. Nos. 5,214,036, 5,707,608 and5,798,349. Liposomal formulations of green porphyrin can be obtainedfrom QLT Inc., Vancouver, Canada. It is contemplated that certain otherphotosensitizers may likewise be formulated with lipid carriers, forexample, liposomes or LDL, to deliver the photosensitizer to CNV.

Furthermore, the photosensitizer can be coupled or conjugated to atargeting molecule that targets the photosensitizer to CNV. For example,the photosensitizer may be coupled or conjugated to a specific bindingligand that binds preferentially to a cell surface component of the CNV,for example, neovascular endothelial homing motif. It appears that avariety of cell surface ligands are expressed at higher levels in newblood vessels relative to other cells or tissues.

Endothelial cells in new blood vessels express several proteins that areabsent or barely detectable in established blood vessels (Folkman (1995)Nature Medicine 1:27-31), and include integrins (Brooks et al. (1994)Science 264: 569-571; Friedlander et al. (1995) Science 270: 1500-1502)and receptors for certain angiogenic factors like vascular endothelialgrowth factor (VEGF). In vivo selection of phage peptide libraries havealso identified peptides expressed by the vasculature that areorgan-specific, implying that many tissues have vascular “addresses”(Pasqualini et al. (1996) Nature 380: 364-366). It is contemplated thata suitable targeting moiety can direct a photosensitizer to the CNVendothelium thereby increasing the efficacy and lowering the toxicity ofPDT.

Several targeting molecules may be used to target photosensitizers tothe neovascular endothelium. For example, α-ν integrins, in particularα-ν β3 and α-ν β5, appear to be expressed in ocular neovascular tissue,in both clinical specimens and experimental models (Corjay et al. (1997)Invest. Ophthalmol. Vis. Sci. 38, S965; Friedlander et al. (1995)supra). Accordingly, molecules that preferentially bind α-ν integrinscan be used to target the photosensitizer to CNV. For example, cyclicpeptide antagonists of these integrins have been used to inhibitneovascularization in experimental models (Friedlander et al. (1996)Proc. Natl. Acad. Sci. USA 93:9764-9769). A peptide motif having anamino acid sequence, in an N- to C-terminal direction, ACDCRGDCFC (SEQID NO: 1)—also know as RGD-4C—has been identified that selectively bindsto human α-ν integrins and accumulates in tumor neovasculature moreeffectively than other angiogenesis targeting peptides (Arap et al.(1998) Nature 279:377-380; Ellerby et al. (1999) Nature Medicine 5:1032-1038). Angiostatin may also be used as a targeting molecule for thephotosensitizer. Studies have shown, for example, that angiostatin bindsspecifically to ATP synthase disposed on the surface of humanendothelial cells (Moser et al. (1999) Proc. Natl. Acad. Sci. USA96:2811-2816).

Clinical and experimental evidence strongly supports a role for vascularendothelial growth factor (VEGF) in ocular neovascularization,particularly ischemia-associated neovascularization (Adamis et al.(1996) Arch. Ophthalmol. 114:66-71; Tolentino et al. (1996) Arch.Ophthalmol. 114:964-970; Tolentino et al. (1996) Ophthalmology103:1820-1828). Potential targeting molecules include antibodies thatbind specifically to either VEGF or the VEGF receptor (VEGF-2R).Antibodies to the VEGF receptor (VEGFR-2 also known as KDR) may alsobind preferentially to neovascular endothelium.

The targeting molecule may be synthesized using methodologies known andused in the art. For example, proteins and peptides may be synthesizedusing conventional synthetic peptide chemistries or expressed asrecombinant proteins or peptides in a recombinant expression system(see, for example, “Molecular Cloning” Sambrook et al. eds, Cold SpringHarbor Laboratories). Similarly, antibodies may be prepared and purifiedusing conventional methodologies, for example, as described in“Practical Immunology”, Butt, W. R. ed., 1984 Marcel Deckker, New Yorkand “Antibodies, A Laboratory Approach” Harlow et al., eds. (1988), ColdSpring Harbor Press. Once created, the targeting agent may be coupled orconjugated to the photosensitizer using standard coupling chemistries,using, for example, conventional cross linking reagents, for example,heterobifunctional cross linking reagents available, for example, fromPierce, Rockford, Ill.

Once formulated, the photosensitizer may be administered in any of awide variety of ways, for example, orally, parenterally, or rectally.Parenteral administration, such as intravenous, intramuscular, orsubcutaneous, is preferred. Intravenous injection is especiallypreferred. The dose of photosensitizer can vary widely depending on thetissue to be treated; the physical delivery system in which it iscarried, such as in the form of liposomes; or whether it is coupled to atarget-specific ligand, such as an antibody or an immunologically activefragment.

It should be noted that the various parameters used for effective,selective photodynamic therapy in the invention are interrelated.Therefore, the dose should also be adjusted with respect to otherparameters, for example, fluence, irradiance, duration of the light usedin PDT, and time interval between administration of the dose and thetherapeutic irradiation. All of these parameters should be adjusted toproduce significant damage to CNV without significant damage to thesurrounding tissue.

Typically, the dose of photosensitizer used is within the range of fromabout 0.1 to about 20 mg/kg, preferably from about 0.15 to about 5.0mg/kg, and even more preferably from about 0.25 to about 2.0 mg/kg.Furthermore, as the dosage of photosensitizer is reduced, for example,from about 2 to about 1 mg/kg in the case of green porphyrin or BPD-MA,the fluence required to close CNV may increase, for example, from about50 to about 100 Joules/cm². Similar trends may be observed with theother photosensitizers discussed herein.

After the photosensitizer has been administered, the CNV is irradiatedat a wavelength typically around the maximum absorbance of thephotosensitizer, usually in the range from about 550 nm to about 750 nm.A wavelength in this range is especially preferred for enhancedpenetration into bodily tissues. Preferred wavelengths used for certainphotosensitizers include, for example, about 690 nm for benzoporphyrinderivative mono acid, about 630 nm for hematoporphyrin derivative, about675 nm for chloro-aluminum sulfonated phthalocyanine, about 660 nm fortin ethyl etiopurpurin, about 730 nm for lutetium texaphyrin, about 670nm for ATX-S10(NA), about 665 nm for N-aspartyl chlorin e6, and about650 nm for 5, 10, 15, 20-tetra (m-hydroxyphenyl) chlorin.

As a result of being irradiated, the photosensitizer in its tripletstate is thought to interact with oxygen and other compounds to formreactive intermediates, such as singlet oxygen and reactive oxygenspecies, which can disrupt cellular structures. Possible cellulartargets include the cell membrane, mitochondria, lysosomal membranes,and the nucleus. Evidence from tumor and neovascular models indicatesthat occlusion of the vasculature is a major mechanism of photodynamictherapy, which occurs by damage to the endothelial cells, withsubsequent platelet adhesion, degranulation, and thrombus formation.

The fluence during the irradiating treatment can vary widely, dependingon the type of photosensitizer used, the type of tissue, the depth oftarget tissue, and the amount of overlying fluid or blood. Fluencespreferably vary from about 10 to about 400 Joules/cm² and morepreferably vary from about 50 to about 200 Joules/cm². The irradiancevaries typically from about 50 mW/cm² to about 1800 mW/cm², morepreferably from about 100 mW/cm² to about 900 mW/cm², and mostpreferably in the range from about 150 mW/cm² to about 600 mW/cm². It iscontemplated that for many practical applications, the irradiance willbe within the range of about 300 mW/cm² to about 900 mW/cm². However,the use of higher irradiances may be selected as effective and havingthe advantage of shortening treatment times.

The time of light irradiation after administration of thephotosensitizer may be important as one way of maximizing theselectivity of the treatment, thus minimizing damage to structures otherthan the target tissues. The optimum time following photosensitizeradministration until light treatment can vary widely depending on themode of administration, the form of administration such as in the formof liposomes or as a complex with LDL, and the type of target tissue.For example, benzoporphyrin derivative typically becomes present withinthe target neovasculature within one minute post administration andpersists for about fifty minutes, lutetium texaphyrin typically becomespresent within the target neovasculature within one minute postadministration and persists for about twenty minutes, N-aspartyl chlorine6 typically becomes present within the target neovasculature within oneminute post administration and persists for about twenty minutes, androse bengal typically becomes present in the target vasculature withinone minute post administration and persists for about ten minutes.

Effective vascular closure generally occurs at times in the range ofabout one minute to about three hours following administration of thephotosensitizer. However, as with green porphyrins, it is undesirable toperform the PDT within the first five minutes following administrationto prevent undue damage to retinal vessels still containing relativelyhigh concentrations of photosensitizer.

The efficacy of PDT may be monitored using conventional methodologies,for example, via fundus photography or angiography. Closure can usuallybe observed angiographically by hypofluorescence in the treated areas inthe early angiographic frames. During the later angiographic frames, acorona of hyperfluorescence may begin to appear which then fills thetreated area, possibly representing leakage from the adjacentchoriocapillaris through damaged retinal pigment epithelium in thetreated area. Large retinal vessels in the treated area typicallyperfuse following photodynamic therapy.

Minimal retinal damage is generally found on histopathologic correlationand is dependent on the fluence and the time interval after irradiationthat the photosensitizer is administered. It is contemplated that thechoice of appropriate photosensitizer, dosage, mode of administration,formulation, timing post administration prior to irradiation, andirradiation parameters may be determined empirically.

It is contemplated that a variety of anti-FasL factors may be combinedwith other treatments for treating unwanted CNV. The anti-FasL factorcan synergistically enhance the activity of the treatment, for example,PDT. In addition, the anti-FasL factor can be used to reduce or delaythe recurrence of the condition. The term “anti-FasL factor” isunderstood to mean any molecule, for example, a protein, peptide,nucleic acid (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)),peptidyl nucleic acid, organic compound or inorganic compound, thatdecreases or eliminates the activity of FasL in a mammal. An “effectiveamount” of an anti-FasL factor is an amount of an anti-FasL factorsufficient to decrease or eliminate the activity of FasL. For example,an effective amount of an anti-FasL antibody is an amount sufficient toreduce or eliminate the binding of FasL to its cognate receptor.

FIG. 1 depicts a representative drawing of the FasL apoptotic pathway.An important mediator of apoptosis is the signaling pathway triggered bythe interaction of the receptor Fas with its ligand (FasL). Fas (alsoknown as Apo1/CD95) is a transmembrane death receptor which, uponcrosslinking with its natural ligand, FasL, induces apoptosis in Fasreceptor-bearing cells. Fas is expressed in a wide variety of tissues,such as the liver, ovary, lung and heart, as well as in myeloid andlymphoblastoid cells. Its cytoplasmic domain contains a 68 amino acidmotif that is both necessary and sufficient for the induction ofapoptosis (death domain, DD). Upon cross-linking (by the Fas Ligand orpolyvalent antibodies), Fas recruits the adaptor FADD (Fas-Associatingprotein with a Death Domain), also known as MORTI, which, via itsN-terminal DED then recruits the pro-enzyme form of caspase-8. Theaggregation of Fas, FADD and caspase-8, named the death-inducingsignaling complex (DISC), catalyses the proteolytic autoactivation ofcaspase-8 (induced-proximity model). The resulting subunits p10 and p18are released into the cytoplasm, where they form an enzymatically activecomplex that triggers the downstream apoptotic caspase cascade.

In age-related macular degeneration, retinal pigment epithelial cellsinhibit choroidal vessel growth through Fas/FasL mediated apoptosis ofchoroidal endothelial cells. In murine carcinoma, FasL+T lymphocytessuppress tumor vessel growth. Anti-FasL neutralizing antibody has beenshown to have anti-angiogenic properties in a murine model ofoxygen-induced retinopathy of prematurity. The anti-FasL neutralizingantibody has also been shown to be safe and effective in reducingvascular leakage and endothelial cell damage in a rat model ofstreptozotocin-induced diabetes. In this model, anti-FasL neutralizingantibody also has a neuroprotective effect by reducing the amount of DNAfragmentation, and therefore apoptosis, in the ganglion cell layer, theouter nuclear and RPE layer, and endothelial cell layer of the retina.The amount of caspase activation is also reduced. Administration of theanti-FasL neutralizing antibody protects against apoptosis in numerousmodels of inflammatory diseases and toxic insults. Additionally, miceinjected with an anti-CD95L antibody 30 minutes after induction ofstroke showed a decrease in both infarct volumes and mortality. Thus,the anti-FasL antibody has anti-angiogenic and anti-permeabilityproperties. Moreover, administration of the anti-FasL antibody protectsagainst apoptosis in numerous models of inflammatory diseases and toxicinsults.

Studies utilizing an anti-FasL neutralizing antibody, which wasadministered in a rat model of laser-induced CNV, reduced angiographicleakage and neovascular formation. Furthermore, there is a synergisticeffect with PDT in treating CNV, and it reduces significantly therecurrences of CNV. PDT treatment in the rat model of laser-induced CNVinduces apoptotic cell death selectively in the endothelial and retinalpigment epithelial cells overlying the treated CNV. This is associatedwith activation of mitochondria and of executional caspases thatultimately lead to cell demise. Administration of anti-FasL neutralizingantibody in combination with PDT reduces the activation of themitochondria, reduces the activation of caspases, and, ultimately,decreases the apoptotic death in the endothelial and RPE cell layer.Thus, without being bound to theory, it appears that PDT-related retinalapoptosis occurs through a Fas/FasL mechanism. As a result, theanti-FasL neutralizing antibody can reduce the collateral damage to theretina when given in combination with PDT, for example, by reducingapoptosis of adjacent retinal cells. Furthermore, the combinationtherapy increases the effectiveness of PDT, reduces the recurrence ofCNV, and protects the retina from PDT-induced apoptosis.

Numerous anti-FasL factors are well known and thoroughly documented inthe art. Examples of anti-FasL factors useful in the practice of theinvention, include, for example, an anti-FasL neutralizing antibody(available, for example, from Pharmingen, San Diego, Calif.); peptidesand nucleic acids (for example, anti-FasL aptamers) that bind FasL toprevent or reduce its binding to its cognate receptor; certainantibodies and antigen binding fragments thereof and peptides that bindpreferentially to the Fas receptor; antisense nucleotides (and PNAs) anddouble stranded RNA for RNAi that ultimately reduce or eliminate theproduction of either FasL or the Fas receptor; soluble Fas; solubleFasL; decoy receptor-3 (DcR3) analogues; matrix metalloproteinases(MMPs); vasoactive intestinal peptide (VIP); pituitary adenylatecyclase-activating polypeptide (PACAP); forskolin; combined use ofbenazepril and valsartan; nonpeptidic corticotropin-releasing hormonereceptor type 1 (CRH-R1)-specific antagonists; mimosine; peptides thatproduce a defective Fas-FasL complex; platelet-activating factor (PAF);and endothelin-1 (ET-1). These anti-FasL factors can act as direct orindirect antagonists of FasL activity.

The anti-FasL factor may be synthesized using methodologies known andused in the art. For example, proteins and peptides may be synthesizedand purified using conventional synthetic peptide chemistries andpurification protocols, or expressed as recombinant proteins or peptidesin a recombinant expression system (see, for example, “MolecularCloning” Sambrook et al. eds, Cold Spring Harbor Laboratories).Similarly, antibodies may be prepared and purified using conventionalmethodologies, for example, as described in “Practical Immunology”,Butt, W. R. ed., 1984 Marcel Deckker, New York and “Antibodies, ALaboratory Approach” Harlow et al., eds. (1988), Cold Spring HarborPress.

Antibodies (e.g., monoclonal or polyclonal antibodies) havingsufficiently high binding specificity for the marker or target protein(for example, FasL or its receptor) can be used as anti-FasL factors. Asnoted above, the term “antibody” is understood to mean an intactantibody (for example, a monoclonal or polyclonal antibody); an antigenbinding fragment thereof, for example, an Fv, Fab, Fa′ or (Fa′)₂fragment; or a biosynthetic antibody binding site, for example, an sFv,as described in U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,704,692. Abinding moiety, for example, an antibody, is understood to bindspecifically to the target, for example, FasL or its receptor, when thebinding moiety has a binding affinity for the target greater than about10⁵ M⁻¹, more preferably greater than about 10⁷ M⁻¹.

Antibodies against FasL or its receptor may be generated using standardimmunological procedures well known and described in the art. See, forexample, Practical Immunology, Butt, N. R., ed., Marcel Dekker, NY,1984. Briefly, isolated FasL or its receptor is used to raise antibodiesin a xenogeneic host, such as a mouse, goat or other suitable mammal.The FasL or its receptor is combined with a suitable adjuvant capable ofenhancing antibody production in the host, and injected into the host,for example, by intraperitoneal administration. Any adjuvant suitablefor stimulating the host's immune response may be used. A commonly usedadjuvant is Freund's complete adjuvant (an emulsion comprising killedand dried microbial cells). Where multiple antigen injections aredesired, the subsequent injections may comprise the antigen incombination with an incomplete adjuvant (for example, a cell-freeemulsion).

Polyclonal antibodies may be isolated from the antibody-producing hostby extracting serum containing antibodies to the protein of interest.Monoclonal antibodies may be produced by isolating host cells thatproduce the desired antibody, fusing these cells with myeloma cellsusing standard procedures known in the immunology art, and screening forhybrid cells (hybridomas) that react specifically with the targetprotein and have the desired binding affinity.

Antibody binding domains also may be produced biosynthetically and theamino acid sequence of the binding domain manipulated to enhance bindingaffinity with a preferred epitope on the target protein. Specificantibody methodologies are well understood and described in theliterature. A more detailed description of their preparation can befound, for example, in Practical Immunology, Butt, W. R., ed., MarcelDekker, New York, 1984.

To the extent that the anti-FasL factor is a nucleic acid or peptidylnucleic acid, such compounds may be synthesized by any of the knownchemical oligonucleotide and peptidyl nucleic acid synthesismethodologies known in the art (see, for example, PCT/EP92/20702 andPCT/US94/013523) and used in antisense therapy. Anti-senseoligonucleotide and peptidyl nucleic acid sequences, usually 10 to 100and more preferably 15 to 50 units in length, are capable of hybridizingto a gene and/or mRNA transcript and, therefore, may be used to inhibittranscription and/or translation of a target protein.

Fas or FasL gene expression can be inhibited by using nucleotidesequences complementary to a regulatory region of the Fas or FasL gene(e.g., the Fas or FasL promoter and/or a enhancer) to form triplehelical structures that prevent transcription of the Fas or FasL gene intarget cells. See generally, Helene (1991) ANTICANCER DRUG DES. 6(6):569-84, Helene et al. (1992) ANN. NY ACAD. SCI. 660: 27-36; and Maher(1992) BIOESSAYS 14(12): 807-15. The antisense sequences may be modifiedat a base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, in the case of nucleotide sequences, phosphodiester linkagesmay be replaced by thioester linkages making the resulting moleculesmore resistant to nuclease degradation. Alternatively, the deoxyribosephosphate backbone of the nucleic acid molecules can be modified togenerate peptide nucleic acids (see Hyrup et al. (1996) BIOORG. MED.CHEM. 4(1): 5-23). Peptidyl nucleic acids have been shown to hybridizespecifically to DNA and RNA under conditions of low ionic strength.Furthermore, it is appreciated that the peptidyl nucleic acid sequences,unlike regular nucleic acid sequences, are not susceptible to nucleasedegradation and, therefore, are likely to have greater longevity invivo. Furthermore, it has been found that peptidyl nucleic acidsequences bind complementary single stranded DNA and RNA strands morestrongly than corresponding DNA sequences (PCT/EP92/20702). Similarly,oligoribonucleotide sequences generally are more susceptible toenzymatic attack by ribonucleases than are deoxyribonucleotidesequences, such that oligodeoxyribonucleotides are are likely to havegreater longevity than oligoribonucleotides for in vivo use.

Additionally, RNAi can serve as an anti-FasL factor. To the extent RNAiis used, double stranded RNA (dsRNA) having one strand identical (orsubstantially identical) to the target mRNA (e.g. Fas or FasL mRNA)sequence is introduced to a cell. The dsRNA is cleaved into smallinterfering RNAs (siRNAs) in the cell, and the siRNAs interact with theRNA induced silencing complex to degrade the target mRNA, ultimatelydestroying production of a desired protein (e.g., Fas or FasL).Alternatively, the siRNA can be introduced directly.

Additionally, aptamers can be used as an anti-FasL factor and may targetFas or FasL. Methods for identifying suitable aptamers, for example, viasystemic evolution of ligands by exponential enrichment (SELEX), areknown in the art and are described, for example, in Ruckman el al.(1998) J. Biol. Chem. 273: 20556-20567 and Costantino et al. (1998) J.Pharm. Sci. 87: 1412-1420. Furthermore, to the extent that the anti-FasLfactor is an organic or inorganic compound, such compounds may besynthesized, extracted and/or purified by standard procedures known inthe art.

The type and amount of anti-FasL factor to be administered may dependupon the PDT and cell type to be treated. It is contemplated, however,that optimal anti-FasL factors, modes of administration and dosages maybe determined empirically. The anti-FasL factor may be administered in apharmaceutically acceptable carrier or vehicle so that administrationdoes not otherwise adversely affect the recipient's electrolyte and/orvolume balance. The carrier may comprise, for example, physiologicsaline.

Protein, peptide or nucleic acid based FasL factor inhibitors can beadministered at doses ranging, for example, from about 0.001 to about500 mg/kg, more preferably from about 0.01 to about 250 mg/kg, and mostpreferably from about 0.1 to about 100 mg/kg. With regard tointravitreal administration, the anti-FasL factor, for example,anti-FasL neutralizing antibody, typically is administered periodicallyas boluses at dosages ranging from about 10 μg to about 5 mg/eye andmore preferably from about 100 μg to about 2 mg/eye.

The anti-FasL factor preferably is administered to the mammal prior toPDT, although it may alternatively or additionally be administeredduring and/or after PDT. Accordingly, it is preferable to administer theanti-FasL factor prior to administration of the photosensitizer. Theanti-FasL factor, like the photosensitizer, may be administered in anyone of a wide variety of ways, for example, orally, parenterally, orrectally. However, parenteral administration, such as intravenous,intramuscular, subcutaneous, subtenons, transcleral, and intravitreal,is preferred. Administration may be provided as a periodic bolus (forexample, intravenously or intravitreally) or as continuous infusion froman internal reservoir (for example, from a bioerodable implant disposedat an intra- or extra-ocular location) or from an external reservoir(for example, from an intravenous bag). The anti-FasL factor may beadministered locally, for example, by continuous release from asustained release drug delivery device immobilized to an inner wall ofthe eye or via targeted trans-scleral controlled release into thechoroid (see, PCT/US00/00207), or the anti-FasL factor may beadministered systemically. Additionally, the anti-FasL factor can beadministered as an ointment, encapsulated in microspheres or liposomes,or placed in a device for longer release.

The present invention, therefore, includes the use of an anti-FasLfactor in the preparation of a medicament for treating, preferably by aPDT-based method, an ocular condition, that preferably is associatedwith choroidal neovasculature. The anti-FasL factor may be provided in akit which optionally may comprise a package insert with instructions forhow to treat such a condition. A composition comprising both aphotosensitizer and an anti-FasL factor may be provided for use in thepresent invention. The composition may comprise a pharmaceuticallyacceptable carrier or excipient. Thus, the present invention includes apharmaceutically acceptable composition comprising a photosensitizer andan anti-FasL factor, as well as the composition for use in medicine.More preferably, however, the invention is for use in combinationtherapy, whereby an anti-FasL factor and a photosensitizer areadministered separately. Preferably, the anti-FasL factor isadministered prior to administration of the photosensitizer.Instructions for such administration may be provided with the anti-FasLfactor and/or with the photosensitizer. If desired, the anti-FasL factorand photosensitizer may be provided together in a kit, optionallyincluding a package insert with instructions for use. The anti-FasLfactor and photosensitizer preferably are provided in separatecontainers. For each administration, the anti-FasL factor and/orphotosensitizer may be provided in unit-dosage or multiple-dosage form.Preferred dosages of photosensitizer and anti-FasL factor, however, areas described above.

In addition, the efficacy and selectivity of the PDT method may beenhanced by combining the PDT with an apoptosis-modulating factor. Anapoptosis-modulating factor can be any factor, for example, a protein(for example a growth factor or antibody), peptide, nucleic acid (forexample, an antisense oligonucleotide), peptidyl nucleic acid (forexample, an antisense molecule), organic molecule or inorganic molecule,that induces or represses apoptosis in a particular cell type. Forexample, it may be advantageous to prime the apoptotic machinery of CNVendothelial cells with an inducer of apoptosis prior to PDT so as toincrease their sensitivity to PDT. Endothelial cells primed in thismanner are contemplated to be more susceptible to PDT. This approach mayalso reduce the light dose (fluence) required to achieve CNV closure andthereby decrease the level of damage on surrounding cells such as RPE.Alternatively, the cells outside the CNV may be primed with an arepressor of apoptosis so as to decrease their sensitivity to PDT. Inthis approach, the PDT at a particular fluence can become more selectivefor CNV.

Apoptosis involves the activation of a genetically determined cellsuicide program that results in a morphologically distinct form of celldeath characterized by cell shrinkage, nuclear condensation, DNAfragmentation, membrane reorganization and blebbing (Kerr et al. (1972)Br. J. Cancer 26: 239-257). At the core of this process lies a conservedset of proenzymes, called caspases, and two important members of thisfamily are caspases 3 and 7 (Nicholson et al. (1997) TIBS 22:299-306).Monitoring their activity can be used to assess on-going apoptosis.

It has been suggested that apoptosis is associated with the generationof reactive oxygen species, and that the product of the Bcl-2 geneprotects cells against apoptosis by inhibiting the generation or theaction of the reactive oxygen species (Hockenbery et al. (1993) Cell 75:241-251, Kane et al. (1993) Science 262: 1274-1277, Veis et al. (1993)Cell 75: 229-240, Virgili et al. (1998) Free Radicals Biol. Med. 24:93-101). Bcl-2 belongs to a growing family of apoptosis regulatory geneproducts, which may either be death antagonists (Bcl-2, Bcl-xL.) ordeath agonists (Bax, Bak.) (Kroemer et al. (1997) Nat. Med. 3: 614-620).Control of cell death appears to be regulated by these interactions andby constitutive activities of the various family members (Hockenbery etal. (1993) Cell 75: 241-251). Several apoptotic pathways may coexist inmammalian cells that are preferentially activated in a stimulus-,stage-, context-specific and cell-type manner (Hakem et al. (1998) Cell94: 339-352).

The apoptosis-inducing factor preferably is a protein or peptide capableof inducing apoptosis in cells for example, endothelial cells, disposedin the CNV. One apoptosis inducing peptide comprises an amino sequencehaving, in an N- to C-terminal direction, KLAKLAKKLAKLAK (SEQ ID NO: 2).This peptide reportedly is non-toxic outside cells, but becomes toxicwhen internalized into targeted cells by disrupting mitochondrialmembranes (Ellerby et al. (1999) supra). This sequence may be coupled,either by means of a crosslinking agent or a peptide bond, to atargeting domain, for example, the amino acid sequence known as RGD-4C(Ellerby et al. (1999) supra) that reportedly can direct theapoptosis-inducing peptide to endothelial cells. Otherapoptosis-inducing factors include, for example, constatin (Kamphaus etal. (2000) J. Biol. Chem. 14: 1209-1215), tissue necrosis factor α(Lucas et al. (1998) Blood 92: 4730-4741) including bioactive fragmentsand analogs thereof, cycloheximide (O'Connor et al. (2000) Am. J.Pathol. 156: 393-398), tunicamycin (Martinez et al. (2000) Adv. Exp.Med. Biol. 476: 197-208), and adenosine (Harrington et al. (2000) Am. J.Physiol. Lung Cell Mol. Physiol. 279: 733-742). Furthermore, otherapoptosis-inducing factors may include, for example, anti-sense nucleicacid or peptidyl nucleic acid sequences that reduce or turn off theexpression of one or more of the death antagonists, for example (Bl-2,Bcl-xL). Antisense nucleotides directed against Bel-2 have been shown toreduce the expression of Bcl-2 protein in certain lines together withincreased phototoxicity and susceptibility to apoptosis during PDT(Zhang et al. (1999) Photochem. Photobiol. 69: 582-586). Furthermore, an18mer phosphorothiate oligonucleotide complementary to the first sixcodons of the Bcl-2 open reading frame, and known as G3139, is beingtested in humans as a treatment for non-Hodgkins' lymphoma.

Apoptosis-repressing factors include, survivin, including bioactivefragments and analogs thereof (Papapetropoulos et al. (2000) J. Biol.Chem. 275: 9102-9105), CD39 (Goepfert et al. (2000) Mol. Med. 6:591-603), BDNF (Caffe et al. (2001) Invest. Ophthalmol. Vis. Sci. 42:275-82), FGF2 (Bryckaert et al. (1999) Oncogene 18: 7584-7593), Caspaseinhibitors (Ekert et al. (1999) Cell Death Differ 6: 1081-1068) andpigment epithelium-derived growth factor including bioactive fragmentsand analogs thereof. Furthermore, other apoptosis-repressing factors mayinclude, for example, anti-sense nucleic acid or peptidyl nucleic acidsequences that reduce or turn off the expression of one or more of thedeath agonists, for example (Bax, Bak).

To the extent that the apoptosis-modulating factor is a protein orpeptide, nucleic acid, peptidyl nucleic acid, or organic or inorganiccompound, it may be synthesized and purified by one or more themethodologies described relating to the synthesis of the anti-FasLfactor.

The type and amount of apoptosis-modulating factor to be administeredmay depend upon the PDT and cell type to be treated. It is contemplated,however, that optimal apoptosis-modulating factors, modes ofadministration and dosages may be determined empirically. The apoptosismodulating factor may be administered in a pharmaceutically acceptablecarrier or vehicle so that administration does not otherwise adverselyaffect the recipient's electrolyte and/or volume balance. The carriermay comprise, for example, physiologic saline.

Protein, peptide or nucleic acid based apoptosis modulators can beadministered at doses ranging, for example, from about 0.001 to about500 mg/kg, more preferably from about 0.01 to about 250 mg/kg, and mostpreferably from about 0.1 to about 100 mg/kg. For example, nucleicacid-based apoptosis inducers, for example, G318, may be administered atdoses ranging from about 1 to about 20 mg/kg daily. Furthermore,antibodies may be administered intravenously at doses ranging from about0.1 to about 5 mg/kg once every two to four weeks. With regard tointravitreal administration, the apoptosis modulators, for example,antibodies, may be administered periodically as bolus dosages rangingfrom about 10 μg to about 5 mg/eye and more preferably from about 100 μgto about 2 mg/eye.

The apoptosis-modulating factor preferably is administered to the mammalprior to PDT. Accordingly, it is preferable to administer theapoptosis-modulating factor prior to administration of thephotosensitizer. The apoptosis-modulating factor, like thephotosensitizer and anti-FasL factor, may be administered in any one ofa wide variety of ways, for example, orally, parenterally, or rectally.However, parenteral administration, such as intravenous, intramuscular,subcutaneous, and intravitreal is preferred. Administration may beprovided as a periodic bolus (for example, intravenously orintravitreally) or by continuous infusion from an internal reservoir(for example, bioerodable implant disposed at an intra- or extra-ocularlocation) or an external reservoir (for example, and intravenous bag).The apoptosis modulating factor may be administered locally, forexample, by continuous release from a sustained release drug deliverydevice immobilized to an inner wall of the eye or via targetedtrans-scleral controlled release into the choroid (see, PCT/US00/00207).

The foregoing methods and compositions of the invention are useful intreating unwanted choroidal neovasculature and thereby ameliorate thesymptoms of ocular disorders including, for example, AMD, ocularhistoplasmosis syndrome, pathologic myopia, angioid streaks, idiopathicdisorders, choroiditis, choroidal rupture, overlying choroid nevi, andinflammatory diseases, and it is contemplated that the same methods andcompositions may also be useful in treating other forms of ocularneovasculature. More specifically, the methods and compositions of theinvention may likewise be useful at treating and removing or reducingcorneal neovasculature, iris neovasculature, retinal neovasculature,retinal angiomas and choroidal hemangiomas.

The invention is illustrated further by reference to the followingnon-limiting example.

Example 1 Anti-FasL Factor Enhances PDT

PDT is an effective treatment for CNV but may require multipletreatments to limit vision loss. Preclinical studies have demonstrateddamage to adjacent retinal structures which may accumulate with multipletreatments. The neuroprotective properties of an anti-FasL neutralizingantibody may offer neuroprotection and improve the effectiveness of PDT.To investigate the efficacy of PDT in combination with an anti-FasLneutralizing antibody in a laser injury model of CNV in the rat, thefollowing experiment was undertaken.

Methods Induction of Choroidal Neovascularization

Choroidal neovascular membranes were induced in Brown-Norway rats usingan Argon/dye laser. Briefly, Brown-Norway rats (Charles RiverLaboratory, Wilmington, Mass.) were anesthetized via an intramuscularinjection of 50 mg/kg of ketamine hydrochloride and 10 mg/kg ofxylazine. Pupils were dilated with a topical application of 5%phenylephrine and 0.8% tropicamide. Six laser spots were induced in eacheye using an Argon/dye laser (532 Argon/dye laser, Coherent medicallaser, Santa Clara, Calif.) by a single investigator.

Administration Ofanti-FasL Antibody In Vivo

These rats were continuously administered (via subcutaneous pump) eitheranti-FasL neutralizing antibody or isotype-matched control at a totaldose of about 5 mg/kg. Briefly, to achieve steady drug levels in thecirculation of animals, the anti-FasL antibody (anti-rat antibody MFL4,Armenian hamster IgG, Pharmingen, San Diego, Calif.) or theisotype-matched control antibody (Armenian hamster anti-TNP IgG,Pharmingen, San Diego, Calif.) was administered by slow intraperitonealrelease from osmotic pumps (Alzet, Cupertino, Calif.) instead ofrepeated intraperitoneal injections. Immediately following the inductionof choroidal neovascularization, osmotic pumps (Alzet, Cupertino,Calif.) were implanted. Two hundred microliters of each antibody at aconcentration of 5 mg/ml were inserted in each osmotic pump, whichreleased 0.5 μl/hr for 14 days.

Fluorescein Angiography

Fluorescein angiography was performed 14 days after the initial lasertreatment using a digital fundus camera system (Model TRC 501A, Topcon,Paramus, N.J.) and standard fluorescein filters to determine ifCNVinduction was successful. CNV closure on fluorescein angiograms wasassessed 24 hours and seven days after verterporfin PDT in a maskedfashion using grading standards. Each animal was given a bolus injectionof 1 ml of 1% sodium fluorescein (Akorn Inc, Decatur, Ill.) in salineintraperitoneally, and the timer was started as soon as the fluoresceinbolus was injected. All angiograms were evaluated in masked fashion bytwo independent retina specialists using grading standards and severityof CNV (baseline FAs) and CNV closure (24 hour FAs).

Photodynamic Therapy in Rats

PDT with verteporfin was performed 14 days after CNV induction. Briefly,rats were anesthetized, and verteporfin, at a dose of 3 mg/m², wasinjected into the tail veins of rats immobilized in a stereotacticframe. The body surface area of each rat was determined based on theirweight according to a nomogram developed by Gilpin. One eye of eachanimal was selected, avoiding eyes that had large subretinal hemorrhagesthat the PDT spot could not cover. Fifteen minutes after the injection,laser light at 689 nm was administered through the pupil with a diodelaser (Coherent medical laser, Santa Clara, Calif.) delivered through aslit lamp adaptor (Laserlink, Coherent medical laser, Santa Clara,Calif.). The laser spot size was set at 759 μm on the plane of theretina. The laser had a constant irradiance of 600 mW/cm² and a fluenceof 25 J/cm² which was delivered for 17 or 42 seconds to achieve totalenergy doses of 10 J/cm².

Western Blotting

The levels of protein expression of caspase 3 and caspase 8, Bax, Bid,Bcl-2, and cytochrome c (Pharmacia) were evaluated by Western blotting.Briefly, whole retinae were lysed for 30 minutes on ice in lysis buffer(50 mM Tris-HCl, pH 8, with 120 mM NaCI and 1% NP-40) supplemented withthe Complete-mini mixture of proteinase inhibitors. The samples werecleared by micro-centrifugation (14,000 rpm, 30 minutes, 4° C.) andassessed for protein concentration. Thirty micrograms of protein/samplewere electrophoresed in a 12% sodium dodecyl sulphate(SDS)-polyacrylamide gel (SDS-PAGE) and electroblotted ontonitrocellulose membranes. After a one hour incubation in blockingsolution (20% IgG-free normal horse serum in phosphate-buffered saline(PBS)), the membranes were exposed overnight at 4° C. to the respectiveprimary antibody. Following washing in PBS, the respective secondaryperoxidase-labeled antibody was applied at a 1:10,000 dilution for onehour at room temperature. The proteins were visualized with the enhancedchemiluminescence technique (Amersham Pharmacia Biotech, Piscataway,N.J.).

Enzyme Activity Assays for Caspase 3 and 8

The enzymatic activity of caspases 3 and 8 were detected in retinallysates with the Apo Alert kit (Clontech, Palo Alto, Calif.).

Tunel Staining

Apoptotic cells were analyzed using the TUNEL technique. Briefly, free3′OH DNA termini were labeled using the TUNEL procedure according to themanufacturer's recommendations (Intergen, NY). TUNEL was performed withhorseradish peroxidase detection in sections from formalin-fixed,paraffin embedded retinas. Whole eyes from rats with or withoutlaser-induced CNV treated with the anti-FasL antibody or theisotype-matched control antibody were fixed in 4% paraformaldehydeovernight at 4° C. Then, TUNEL staining was performed.

Statistics

Differences in CNV induction between treatment groups were evaluatedusing chi-square tests. Lesions that did not show significant leakagewere excluded from the statistical analysis. Retinal levels of Bcl-2,Bax, Bcl-xL, and Bid were measured by Western blotting, and cystolic andmitochondrial levels of cytochrome c were measured by a modified ELISAmethod. Activation of caspases-3 and -6 were measured with a modifiedELISA method in whole retinal lysates.

Results Anti-FasL Antibody Treatment Reduces Angiographic Leakage inLaser-Induced CNV

To assess whether the anti-FasL antibody treatment influences theangiographic leakage in the laser-induced CNV model, the percentage oflesions that were closed in rats that received the anti-FasL antibodytreatment was compared to the percentage from those that received thecontrol antibody treatment. The percentage of angiographically “leaky”lesions (grade IIA+IIB) among the anti-FasL antibody treated rats was82% and was 97.4% in the control antibody treated rats (P<0.0001).Whereas, the percentage of the angiographically non-leaky and less leakylesions (grade I+0) was 19% in rats that received the anti-FasL antibodytreatment and 2.6% in the control antibody treated rats (FIG. 6). Thus,FIG. 6 provides evidence that anti-FasL antibody treatment reducesangiographic leakage in laser-induced CNV.

Anti-FasL Antibody Treatment Reduces PDT-Induced Angiographic Leakage inLaser-Induced CNV

To assess whether the anti-FasL treatment influences the angiographicleakage after PDT in the laser induced CNV model, the percentage oflesions that were closed in rats that received PDT and the anti-FasLantibody treatment was compared to the percentage from those thatreceived the control treatment. Because the anti-FasL antibody treatmentreduced the angiographic leakage in a statistically significant mannerrelative to the control antibody, lesions on which to perform PDT wereselected that had an equal degree of angiographic leakage in the twoantibody-treated populations. The percentage of closed lesions among theanti-FasL antibody and PDT treated rats was 100% and was 69% in thecontrol and PDT treated rats (FIG. 5). Thus, FIG. 5 provides evidencethat anti-FasL antibody treatment reduces angiographic leakage afterPDT. Because the anti-FasL antibody had a marginal effect on non-leakylesions as shown by the comparison between laser-induced CNV animalsreceiving anti-FasL antibody treatment and control antibody treatment(19% vs. 2.6%, FIG. 6), the fact that 100% of the animals receivingcombined anti-FasL antibody and PDT treatment showed closed lesions incomparison to 69% of the animals receiving combined control antibody andPDT treatment indicates that the combination produces more than anadditive effect and is synergistic.

PDT Increases the Expression of the Fas Receptor in the Rat Retina

It is understood that treatment with the photosensitizer verteporfinincreases the apoptotic cell death both in vivo and in vitro. It wassuggested that PDT acts in concert with the Fas apoptotic signalingpathway, because a Fas-activating antibody can potentiate thePDT-induced cell death of thymic cells in vivo. This experimentindicated that verteporfin PDT increases the retinal levels of theapoptotic death receptor, Fas, an indication that the Fas/FasL pathwayplays a causative role in the apoptotic retinal cell death after PDT(FIG. 3). Thus, FIG. 3 provides evidence that PDT increases theexpression of the Fas receptor in the rat retina.

Anti-FasL Antibody Treatment Reduces PDT-Induced Apoptosis inLaser-Induced CNV

To investigate the role of Fas/FasL in verteporfin-PDT, the occurrenceof apoptotic cell death in the retinas of rats that had received,concurrent with PDT, the anti-FasL antibody treatment or the controlantibody was studied. It was found that rats that had received thecontrol antibody treatment plus PDT showed apoptotic cells in the RPE,photoreceptor and endothelial cell layer, whereas the anti-FasL antibodytreatment significantly reduced this apoptotic death from PDT. Rats thatreceived only the anti-FasL antibody or control antibody showed aminimal amount of apoptotic death.

Anti-FasL Antibody Treatment Nrevents PDT-Induced Activation of Caspases3 and 8 in Laser-Induced CNV

Fas/FasL mediated apoptotic cell death involves the activation of apical(receptor mediated) and executional caspases. Rats treated with PDT andcontrol antibody showed cleavage of the proform of caspases 3 and 8 totheir respective activated fragments. The immunoblotting results wereconfirmed with caspase activity assays. The anti-FasL antibody inhibitedthe PDT-induced activation of caspases (FIGS. 4 and 7). Thus, FIG. 4provides evidence that anti-FasL antibody treatment prevents activationof caspase 3 after PDT, and FIG. 7 provides evidence that anti-FasLantibody treatment reduces PDT-induced caspase 8 activation inlaser-induced CNV.

Anti-FasL Antibody Treatment Reduces PDT-Induced Bax Upregulation andBcl-2 Downre Ulation in Laser-Induced CNV

Apoptosis and cell survival is the outcome of a delicate balance betweenanti-apoptotic genes, such as Bcl-2, and pro-apoptotic genes, such asBax, which were shown to influence Fas-mediated apoptosis in a varietyof models. In the present animal model, PDT-induced apoptosis isassociated with downregulation of Bcl-2 protein levels and upregulationof Bax protein levels. Treatment with the anti-FasL antibody, but notthe control antibody, attenuates the PDT-induced downregulation of Bcl-2level and upregulation of Bax level (FIGS. 9 and 10). Thus, FIG. 9provides evidence that anti-FasL antibody treatment reduces PDT-inducedBax upregulation in laser-induced CNV, and FIG. 10 provides evidencethat anti-FasL antibody treatment reduces PDT-induced Bcl-2downregulation in laser-induced CNV.

Anti-FasL Antibody Treatment Prevents PDT-Induced Cleavage of Bid inLaser-Induced CNV

In the present animal model, PDT-induced apoptosis is associated withcleavage of Bid. Treatment with the anti-FasL antibody, but not thecontrol antibody, attenuates the PDT-induced cleavage of Bid (FIG. 2).Thus, FIG. 2 provides evidence that anti-FasL antibody treatmentprevents cleavage of Bid after PDT.

Anti-FasL Antibody Treatment Reduces PDT-Induced Cytochrome c Release inLaser-Induced CNV

In the present animal model, PDT-induced apoptosis is associated withrelease of mitochondrial cytochrome c into the cytoplasm. Treatment withthe anti-FasL antibody, but not the control antibody, attenuates thePDT-induced release of mitochondrial cytochrome c into the cytoplasm(FIG. 8). Thus, FIG. 8 provides evidence that anti-FasL antibodytreatment reduces PDT-induced cytochrome c release in laser-induced CNV.

Discussion

In the present study, the efficacy of PDT in combination with a FasLneutralizing antibody was investigated. It was found that continuoussubcutaneous administration of the anti-FasL antibody, but not theisotype matched control antibody, reduced the angiographic leakage fromCNV and increased the efficacy of verteporfin PDT on CNV closure.

These findings suggest that apoptotic mechanisms may participate inretinal cell loss during PDT. The involvement of the Fas/FasLreceptor/ligand pair and the Bcl-2 family members were also investigatedin this model. The Bcl-2 family includes several anti-apoptotic members,such as Bcl-2 and Bcl-xL, whereas Bax and the cleaved form of Bidpromote apoptosis. The balance between pro- and anti-apoptotic membersof the Bcl-2 family regulates the fate of mitochondrial cytochrome c.When the pro-apoptotic stimuli predominate, cytochrome c moves from themitochondria to the cytoplasm, where, together with Apafl, it activatespro-caspase-9. The active form of caspase-9 then activates theexecutioner caspases-3 and -6. PDT decreased the retinal levels ofBcil-2 and Bcl-xL, increased the levels of Bax, and induced Bidcleavage. The release of cytochrome c to the cytosol and activation ofcaspases-3 and -6 also were detected.

Treatment with the anti-FasL neutralizing antibody, but not the isotypematched control antibody, reversed the above-mentioned changes. Thissuggests a role for the Fas/FasL pathway in triggering this apoptoticcascade. The Fas receptor can activate caspase-9 and, subsequently,caspases-3 and -6, via two pathways. The first pathway is activation ofcaspase-8, which can directly cleave and activate caspase-9 (type Ipathway). The second pathway is, when the level of caspase-8 activationis insufficient to cleave caspase-9 due to low endogenous pro-caspase-8expression or due to the presence of caspase-8 inhibitors such as FLIP,amplification of the apoptotic signal of caspase-8 via the mitochondria(type II pathway). Specifically, caspase-8 cleaves Bid into its active,pro-apoptotic form. This then triggers cytochrome c release from themitochondria and the above-mentioned activation of caspase-9 and thedownstream executioner caspases (See FIG. 1). The ability of a Fas/FasLneutralizing agent to inhibit the downstream apoptotic signaling pathwayin this study confirms its role in triggering retinal cell apoptosis inPDT.

The ability of a Fas/FasL inhibitor to suppress retinal cell apoptosisin PDT-treated retinae may also have therapeutic implications.Concurrent treatment with a Fas/FasL neutralizing agent, such as ananti-Fas or anti-FasL antibody, or possibly with a caspase inhibitor,may limit the damage to adjacent normal structures that occurs duringPDT. This may allow more intensive PDT treatments, thus potentiallyimproving PDT results. Due to the involvement of the Fas apoptoticpathway in several other disease models, such as hepatitis, there isstrong interest in drug development of Fas/FasL inhibitors that would beeffective and safe for human use.

In conclusion, continuous subcutaneous administration of the anti-FasLneutralizing antibody, and not the isotype matched control antibody,reduced the angiographic leakage from CNV (97.4% of the lesions in theanimals treated with the control antibody were stage IIA and IIB versus82% in the anti-FasL antibody treated group, P<0.001) and increased theefficacy of verteporfin PDT on CNV closure (69% of the lesions in theanimals treated with the control antibody and PDT were angiographicallynot perfused versus 100% of lesions treated with anti-FasL antibody andPDT). PDT decreased the retinal levels of Bcl-2 and Bcl-xL, increasedthe levels of Bax, and induced cleavage of Bid and release of cytochromec into the cytosol. PDT also induced activation of caspases-3 and -6 inthe retina. Treatment with the anti-FasL neutralizing antibody, but notthe isotype-matched control, reversed these changes.

Anti-FasL antibody administration decreased the agiographic leakage,increased the efficacy of Verteporfin PDT for CNV closure in a ratmodel, and reduced the collateral apoptotic damage induced by PDT. Thissuggests that the combination of PDT with anti-FasL neutralizing agents(i.e., anti-FasL factors) may limit damage to normal structures andimprove PDT results.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificpublications disclosed hereinabove is expressly incorporated herein byreference for all purposes.

1-16. (canceled)
 17. A method for reducing photoreceptor cell deathassociated with a photodynamic-therapy-based method that uses aphotosensitizer to treat unwanted choroidal neovasculature in a mammal,the method comprising administering to an eye of the mammal an amount ofan anti-FasL factor sufficient to reduce photoreceptor cell deathassociated with photodynamic therapy relative to photodynamic therapywithout the anti-FasL factor, wherein the anti-FasL factor is selectedfrom the group consisting of an anti-FasL antibody, an anti-Fasantibody, decoy receptor 3, and a decoy receptor 3 analog.
 18. Themethod of claim 17, wherein the mammal is a primate.
 19. The method ofclaim 18, wherein the primate is human.
 20. The method of claim 17,wherein the photosensitizer is an amino acid derivative, an azo dye, axanthene derivative, a chlorin, a tetrapyrrole derivative, or aphthalocyanine.
 21. The method of claim 20, wherein the photosensitizeris lutetium texaphyrin, a benzoporphyrin, a benzoporphyrin derivative, ahematoporphyrin, or a hematoporphyrin derivative.
 22. The method ofclaim 21, wherein the photosensitizer is lutetium texaphyrin.
 23. Themethod of claim 21, wherein the photosensitizer is a benzoporphyrin. 24.The method of claim 21, wherein the photosensitizer is a benzoporphyrinderivative.
 25. The method of claim 17, wherein the anti-FasL factor isan anti-FasL antibody.
 26. The method of claim 17, wherein the anti-FasLfactor is an anti-Fas antibody.
 27. The method of claim 17, wherein theanti-FasL factor is decoy receptor-3.
 28. The method of claim 17,wherein the anti-FasL factor is a decoy receptor-3 analog.
 29. Themethod of claim 17, wherein the anti-FasL factor is administered to themammal prior to photodynamic therapy.
 30. The method of claim 17,wherein the anti-FasL factor is administered to the mammal duringphotodynamic therapy.
 31. The method of claim 17, wherein the methodameliorates the symptoms of age-related macular degeneration.
 32. Themethod of claim 17, wherein the photoreceptor cell death is apoptoticcell death.